Follicle-stimulating hormone (simply referred to as FSH) is a commonly used major drug ingredient in the field of animal breeding. The existing marketed FSH is mainly a biochemical species extracted from porcine pituitary and is applicable for early estrus and synchronous estrus of young sows, as well as for estrus stimulation of sows having delay in their estrus. It also has broader application in the field of the breeding of cattle and sheep. Biochemically extracted FSH has the defects such as virus contamination, limited sources of raw materials, collection difficulties, low content, and complex purification process. In addition, due to the limitations of the detection methods and the virus inactivation technology as well as the occurrence of unpredictable infection of new pathogenic virus, the possibility of the virus contamination of the biochemically extracted products cannot be completely eliminated.
In contrast, recombinant FSH has advantages that are unmatchable by biochemical FSH products in terms of purity, antigenicity, safety, and absence of viral infection. To date, no recombinant FSH product has been used in the field of veterinary medication. FSH is a glycosylated protein whose molecular weight is 43 KD as measured by SDS-PAGE. In addition, hFSH is a glycosylated protein comprising two single chains (an α chain and a β chain) connected by non-covalent bonds, and the correct folding of the two chains can ensure the bioactivity of hFSH. It remains a challenge to maintain the normal binding of the two chains during the expression and purification process of the protein. As a therapeutic drug, in order to ensure its biological activity, a necessary condition is to have the correct three-dimensional structure and glycosylation modification. Having perfect post-translational modification function is the main reason why mammalian cells are selected as the expression hosts of most biopharmaceutical proteins. Among all mammalian cells, Chinese Hamster Ovary Cell (CHO) is the most successful host cell for the expression of eukaryotic heterologous genes. More and more pharmaceutical recombinant proteins have achieved high-efficient expression in CHO cells, and many recombinant protein drugs for human use have already been marketed. Compared with other expression systems, this system has many advantages, such as having a complete post-translational processing process, including glycosylation and hydroxylation, so that the expression products of the heterologous eukaryotic genes can maintain their natural structure and activity, and the expression products are secreted extracellularly, which is favorable for the separation and purification of the exogenous proteins.
The amino acid sequence homology of FSH proteins between different mammalian species is very high. For example, the amino acid sequence homology of the α chain and β chain of human FSH to the α chain and β chain of porcine FSH are 83% and 96%, respectively, while the amino acid sequence homology of human FSH to bovine FSH is as high as 88%, suggesting the potential application of hFSH in the field of the breeding of other mammalian animals. Currently, pharmaceutical recombinant hFSH (human follicle-stimulating hormone, simply referred to as hFSH) produced by using CHO cells has been marketed. However, the following problems still exist. First, the expression amount of the recombinant hFSH produced by the existing methods is too low, its preparation process is complicated, and the production cost is too high. Secondly, the half-life thereof is short, requiring frequent administration by injection. Therefore, it is a challenge in this field to take advantage of molecular biology and cell culture method to develop hFSH drugs with biological activity and longer half-life.